Shahid Ullah (Presenter)
Karolinska Institute
Authorship: Shahid Ullah (1), Olof Beck (2)
(1) Karolinska Institute, (2) Karolinska Institute & Karolinska University Hospital
| Short Abstract Drugs abusing is a serious public health concern that affects almost every community in the world. Using these substances may lead to a severe mental and physical disorder and may endanger human life. In this study, a mass spectrometric method has been validated to analyze 29 psychoactive drugs in exhaled breath. Approximately 30L of exhaled breath was sampled to a polymer filter, extracted with methanol and analyzed by LC-MS/MS. Method detection limits were ≤ 10 pg/filter for most of the analytes and method recoveries were in the range of 70 to 120% for all analytes. |
Long Abstract
Introduction
Substance abuse is a serious public health concern that affects almost every society in the world. Using these substances may lead to a severe mental and physical disorder and may endanger human life. It has been a common laboratory investigation in clinical and forensic applications for testing drugs of abuse. Most typical methodology includes blood and urine testing for investigating these substances. These procedures may often need proper supervision to reduce the inconvenience of the donor and prevent any intrusion of the sampling. Recently, an alternative matrix, exhaled breath, gained research of interest in the clinical lab for investigating the suspect of drugs of abuse. A simple collection device is now available to collect exhaled particles which proved to be convenient for the patient. This study describes a LC-MS method which enables to analyze 29 drugs of abuse in exhaled particles collecting by a suitable collection procedure.
Methodology
Particles in exhaled breath was collected in a commercial sampling. A total of 30 L exhaled air pass through the device. During sampling of exhaled breath containing microparticles pass through a mouth-piece and subsequently collected to a polymer filter with a diameter of 30 mm inside the device. During extraction, 5 ml MeOH was used to extract the analyte from the filter. The extracts were evaporated at 40 °C with vacuum centrifugation. Afterwards, residuals were reconstituted with 70 μL of MeOH:H2O containg 0.1% NH3 (1:1). Samples were analyzed by HPLC-MS/MS using a Dionex Ultimate 3000 UHPLC system coupled to a Thermo Fisher Scientific TSQ Quantiva triple quadropole mass spectrometer. The chromatographic separation was achieved on an acquity UPLC BEH phenyl column (2.1 × 100 mm, 1.7 µm) with injection volume of 2 µL at a mobile phase flow rate 450 µl/min in a gradient mode with column oven temperature at 50 °C. Mobile phase A consisting of 95:5 water:methanol and a mobile phase B consisting of 95:5 MeOH: water, with 4 mM ammonium formate and 0.2% ammonia in both A and B. The gradient profile was as follows: starting with 30% B (hold time 0.4 min) and continued with linear change to 55% B up to 1 min and to 98% B up to 3.0 min. Continued 98% B up to 4.45 min and returned to initial condition at 4.5 min followed by equilibration until 5 min. The mass spectrometer was operated in electrospray ionization in positive ion mode. Ion spray voltage was 2.7 kV, source and ion transfer tube temperature was 450 and 280 °C, respectively. Two SRM transitions were used for each compound during acquisition. Method was validated by assigning method detection and quantification limits, linearity, recovery, matrix effect, precision and accuracy, and method application.
Results
Chromatographic separation was performed based on a previously developed method in our lab with some modification. Excellent chromatographic separation was achieved with the column mentioned above. The method detection limits (MDLs) were in the range of 0.3 to 5 pg/filter and 10 to 50 pg/filter for 21 (out of 29) and 8 compounds, respectively. The whole method linearity was assessed by making a 6 point standard calibration curve with native analytes spiking the polymer filter with concentrations ranging from individual MLQs to 15 ng/filter. Excellent method linearity was achieved (r2 ≥ 0.99) for all analytes indicating that the recoveries were not concentration dependent within the concentration range. The method recovery was in the range of 70 – 120% for all analytes. Other validation parameters including accuracy and precision, matrix effect, carry over and interference were achieved with satisfactory performance. Finally, for the method application, breath samples from intoxicated patient will be collected from Stockholm alcohol and drug rehabilitation centre and results will be presented later.
Conclusions.
In this study, we successfully validated an analytical method based on chromatographic separation and mass spectrometric analyzer for analyzing drugs of abuse in human breath. Excellent results were achieved for all validation parameters including method detection limits as low as pg level for most of the compounds. Total 29 drugs of abuse will be analyzed in intoxicated patient samples.
References & Acknowledgements:
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